Factor VII (FVII) is a vitamin K dependent glycoprotein which in its activated form (FVIIa) takes part in the coagulation process activating the Factor X and the Factor IX in the presence of calcium and of tissue factor. FVII is secreted in form of a single peptide chain of 406 residues, with a molecular weight of about 50 kDa. The FVII contains four distinctive structural domains: the N-terminal gamma-carboxylic domain (Gla), two “epidermal growth factor (EGF)-like” domains, and a serine protease domain. The activation of the FVII into FVIIa is characterized by the cleavage of the Arg152-Ile153 domain (Arginine 152-Isoleucine 153) linkage. The FVIIa is, therefore, a compound with a light chain of 152 amino acids with a molecular weight of about 20 kDa, and with a heavy chain of 254 aminoacids with a molecular weight of about 30 kDa linked one to another with a single disulfide bridge (Cys135-Cys262).
The plasma FVIIa contains several post-translational modifications: the first ten glutamic acids are [gamma]-carboxylated, Asp63 is partially hydroxylated, Ser52 (Serine 52) and Ser60 (Serine 60) are 0-glycosylated and carry the Glucose (Xylose)0-2 and Fucose patterns, respectively, Asn145 (Asparagine 145) and Asn322 (Asparagine 322) are N-glycosylated mainly by biantennary bisialylated complex structures.
FVII is used for the treatment of patients suffering from hemophilia, showing a deficiency of Factor VIII (type A hemophilia) or of Factor IX (type B hemophilia), as of patients showing other deficiencies of coagulation factors, for example, a congenital deficiency of FVII. It is, therefore, necessary that concentrates of injectable FVIIa be available.
The most ancient method for obtaining FVIIa concentrates consisted in the purification of FVIIa from plasma proteins resulting from plasma fractionation. For that purpose, EP 0 346 241 describes the preparation of a FVIIa-enriched fraction, obtained after adsorption and then elution of a secondary product of the fractionation of plasma proteins containing FVII and FVIIa and other proteins such as Factors IX (FIX), X (FX) and II (FII), particularly the pre-eluate of PPSB (P=prothrombin or FII, P=proconvertin or FVII, S=Stuart Factor or FX and B=antihemophilic Factor B or FIX). The drawback of this process is that the obtained FVII still contains some traces of the other coagulation factors.
Likewise, document EP 0 547 932 describes a manufacturing process of a high purity FVIIa concentrate substantially free of vitamin-K-dependent factors and of FVIII. The FVII obtained by this process, in spite of its purity, shows a residual thrombogenic activity.
Generally speaking, one of major drawbacks of these processes is that they yield only small amounts of products. It is, moreover, still difficult to obtain a product entirely free of other proteins present in the plasma. Finally, although a number of precautions are being implemented at every stage of the preparation of plasma coagulation factors in order to ensure their viral and bacterial safety (follow-up of blood donors, tests for detecting of known viral and bacterial contaminants, stringent purification and virus inactivating treatments in order to reduce as far as possible the hazard of transmission of blood-born pathogenic agents), nevertheless, all risks of contamination with pathogenic agents are not excluded. In addition, the appearance of a new variant of the Creutzfeldt-Jakob disease gave rise to fears of transmission of unconventional pathogenic agents by blood products. Moreover, the volume of plasma collected from donors remains limited.
Therefore, since the 1980s, the DNA encoding the human Factor VII was isolated (Hagen et al. Proc. Natl. Acad. Sci., 1986, 83(8):2412-6) and expressed in various expression systems.
Various processes for the purification of recombinant Factor VII have further been described (see e.g. WO2009/141418). Currently recombinant Factor VII polypeptides are typically purified by one or two different multi-step processes, either employing exclusively chromatography on conventional resins, or including affinity chromatography employing an affinity resin with protein ligands.
Recombinant Factor VII may be produced from fermentation batches, or from transgenic mammals. In particular compositions of FVII produced in the mammary glands of female rabbit are described, e.g. in patent application WO2007/138199. The FVII produced in the milk by transgenic mammals can then be purified from milk e.g. by tangential filtration and chromatography, or by calcium precipitation followed by several chromatography steps.
However the available purification methods are not devoid of drawbacks. Especially native proteins from the transgenic mammal, and in particular native Factor VII from the transgenic mammal, might still be present, which would trigger immunogenicity upon administration to a human patient.